Sustained Effects of CGRP Blockade on Cortical Spreading Depolarization-Induced Alterations in Facial Heat Pain Threshold, Light Aversiveness, and Locomotive Activity in the Light Environment.

A migraine is clinically characterized by repeated headache attacks that entail considerable disability. Many patients with migraines experience postdrome, the symptoms of which include tiredness and photophobia. Calcitonin gene-related peptide (GGRP) is critically implicated in migraine pathogenesis. Cortical spreading depolarization (CSD), the biological correlate of migraine aura, sensitizes the trigeminovascular system. In our previous study, CSD caused hypomotility in the light zone and tendency for photophobia at 72 h, at which time trigeminal sensitization had disappeared. We proposed that this CSD-induced disease state would be useful for exploring therapeutic strategies for migraine postdrome. In the present study, we observed that the CGRP receptor antagonist, olcegepant, prevented the hypomotility in the light zone and ameliorated light tolerability at 72 h after CSD induction. Moreover, olcegepant treatment significantly elevated the threshold for facial heat pain at 72 h after CSD. Our results raise the possibility that CGRP blockade may be efficacious in improving hypoactivity in the light environment by enhancing light tolerability during migraine postdrome. Moreover, our data suggest that the CGRP pathway may lower the facial heat pain threshold even in the absence of overt trigeminal sensitization, which provides an important clue to the potential mechanism whereby CGRP blockade confers migraine prophylaxis.

Protracted hypomobility in the absence of trigeminal sensitization after cortical spreading depolarization: Relevance to migraine postdrome.

Migraine sufferers often exhibit photophobia and physical hypoactivity in the postdrome and interictal periods, for which no effective therapy currently exists. Cortical spreading depolarization (CSD) is a neural phenomenon underlying migraine aura. We previously reported that CSD induced trigeminal sensitization, photophobia, and hypomobility at 24 h in mice. Here, we examined the effects of CSD induction on light sensitivity and physical activity in mice at 48 h and 72 h. Trigeminal sensitization was absent at both time points. CSD-subjected mice exhibited significantly less ambulatory time in both light (P = 0.0074, the Bonferroni test) and dark (P = 0.0354, the Bonferroni test) zones than sham-operated mice at 72 h. CSD-subjected mice also exhibited a significantly shorter ambulatory distance in the light zone at 72 h than sham-operated mice (P = 0.0151, the Bonferroni test). Neurotropin® is used for the management of chronic pain disorders, mainly in Asian countries. The CSD-induced reductions in ambulatory time and distance in the light zone at 72 h were reversed by Neurotropin® at 0.27 NU/kg. Our experimental model seems to recapitulate migraine-associated clinical features observed in the postdrome and interictal periods. Moreover, Neurotropin® may be effective in ameliorating postdromal/interictal hypoactivity, especially in a light environment.

Bilateral pneumothorax after acupuncture treatment.

A 31-year-old female physician was diagnosed with bilateral pneumothorax a day after her acupuncture treatment. Her body mass index was 16.9 and she did not have a prior history of respiratory disease or smoking. Acupuncture needles may easily reach the pleura around the end of the suprascapular angle of the levator scapulae muscle where the subcutaneous tissue is anatomically thin. In our patient, the thickness between the epidermis and the visceral pleura in this area was only 22 mm as confirmed by an ultrasound scan. Although she felt chest discomfort 30 min after the procedure, she assumed the symptom to be a reaction to the acupuncture. In light of our case, we advise practitioners to select appropriate acupuncture needles for patients based on the site of insertion and counsel them regarding the appearance of symptoms such as chest pain and dyspnoea immediately after the procedure.

Resilience to capsaicin-induced mitochondrial damage in trigeminal ganglion neurons.

Capsaicin is an agonist of transient receptor potential cation channel subfamily V member 1 (TRPV1). Strong TRPV1 stimulation with capsaicin causes mitochondrial damage in primary sensory neurons. However, the effect of repetitive and moderate exposure to capsaicin on the integrity of neuronal mitochondria remains largely unknown. Our electron microscopic analysis revealed that repetitive stimulation of the facial skin of mice with 10 mM capsaicin induced short-term damage to the mitochondria in small-sized trigeminal ganglion neurons. Further, capsaicin-treated mice exhibited decreased sensitivity to noxious heat stimulation, indicating TRPV1 dysfunction, in parallel with the mitochondrial damage in the trigeminal ganglion neurons. To analyze the capsaicin-induced mitochondrial damage and its relevant cellular events in detail, we performed cell-based assays using TRPV1-expressing PC12 cells. Dose-dependent capsaicin-mediated mitochondrial toxicity was observed. High doses of capsaicin caused rapid destruction of mitochondrial internal structure, while low doses induced mitochondrial swelling. Further, capsaicin induced a dose-dependent loss of mitochondria and autophagy-mediated degradation of mitochondria (mitophagy). Concomitantly, transcriptional upregulation of mitochondrial proteins, cytochrome c oxidase subunit IV, Mic60/Mitofilin, and voltage-dependent anion channel 1 was observed, which implied induction of mitochondrial biogenesis to compensate for the loss of mitochondria. Collectively, although trigeminal ganglion neurons transiently exhibit mitochondrial damage and TRPV1 dysfunction following moderate capsaicin exposure, they appear to be resilient to such a challenge. Our in vitro data show a dose-response relationship in capsaicin-mediated mitochondrial toxicity. We postulate that induction of mitophagy and mitochondrial biogenesis in response to capsaicin stimulation play important roles in repairing the damaged mitochondrial system.

Cortical spreading depolarisation-induced facial hyperalgesia, photophobia and hypomotility are ameliorated by sumatriptan and olcegepant.

Cortical spreading depolarisation (CSD), the neural mechanism underlying migraine aura, may cause headache by sensitising the trigeminal system. Photophobia, the most bothersome accompanying symptom during migraine attacks, is more prevalent in migraine with aura than in migraine without aura. Whether CSD plays a role in developing photophobia remains unknown. Moreover, migraine-induced physical hypoactivity contributes to loss of productivity. We aimed to investigate the development of trigeminal sensitisation, photophobia and locomotive abnormality after KCl-induced CSD using 86 male C57BL/6 mice. Sham-operated mice were used as controls. We confirmed the presence of trigeminal sensitisation and photophobia at 24 h after CSD. CSD-subjected mice also exhibited significantly reduced locomotive activity in both light and dark zones. Hence, the CSD-induced hypomobility was likely to be independent of photophobia. The 5-HT1B/1D agonist, sumatriptan, corrected all these CSD-induced abnormalities. Moreover, dose dependency was demonstrated in the ameliorating effect of the calcitonin gene-related peptide (CGRP) receptor antagonist, olcegepant, on these abnormalities. Sumatriptan and olcegepant improved mouse locomotion with therapeutic lags ranging from 20 to 30 min. Collectively, CSD caused trigeminal sensitisation, photophobia and hypomobility that persisted for at least 24 h by a mechanism involving the 5-HT1B/1D and CGRP activity.

Functional interactions between transient receptor potential M8 and transient receptor potential V1 in the trigeminal system: Relevance to migraine pathophysiology.

Background Recent genome-wide association studies have identified transient receptor potential M8 ( TRPM8) as a migraine susceptibility gene. TRPM8 is a nonselective cation channel that mediates cool perception. However, its precise role in migraine pathophysiology is elusive. Transient receptor potential V1 (TRPV1) is a nonselective cation channel activated by noxious heat. Both TRPM8 and TRPV1 are expressed in trigeminal ganglion (TG) neurons. Methods We investigated the functional roles of TRPM8 and TRPV1 in a meningeal inflammation-based migraine model by measuring the effects of facial TRPM8 activation on thermal allodynia and assessing receptor coexpression changes in TG neurons. We performed retrograde tracer labeling to identify TG neurons innervating the face and dura. Results We found that pharmacological TRPM8 activation reversed the meningeal inflammation-induced lowering of the facial heat pain threshold, an effect abolished by genetic ablation of TRPM8. No significant changes in the heat pain threshold were seen in sham-operated animals. Meningeal inflammation caused dynamic alterations in TRPM8/TRPV1 coexpression patterns in TG neurons, and colocalization was most pronounced when the ameliorating effect of TRPM8 activation on thermal allodynia was maximal. Our tracer assay disclosed the presence of dura-innervating TG neurons sending collaterals to the face. Approximately half of them were TRPV1-positive. We also demonstrated functional inhibition of TRPV1 by TRPM8 in a cell-based assay using c-Jun N-terminal kinase phosphorylation as a surrogate marker. Conclusions Our findings provide a plausible mechanism to explain how facial TRPM8 activation can relieve migraine by suppressing TRPV1 activity. Facial TRPM8 appears to be a promising therapeutic target for migraine.

Signaling Pathways Relevant to Nerve Growth Factor-induced Upregulation of Transient Receptor Potential M8 Expression.

Transient receptor potential melastatin 8 (TRPM8) is a nonselective cation channel that primarily detects the innocuous cold. In pathological conditions, TRPM8 plays a role in the development of cold hyperalgesia/allodynia. Nerve growth factor (NGF) is an important mediator involved in various pain disorders. In the present study, the NGF-TrkA pathway increased TRPM8 expression by stabilizing TRPM8 mRNA through the actions of phosphatidylinositol 3-kinase and p38 MAP kinase. Moreover, c-Jun N-terminal kinase and Src tyrosine kinase were identified as a positive and negative regulator of TRPM8 expression, respectively, via post-transcriptional mechanisms independent of mRNA stabilization. PTEN activity was found to increase protein TRPM8 expression. Calcium imaging confirmed that NGF induced TRPM8 functional upregulation. Time-lapse fluorescence microscopic analysis and a cell fractionation assay revealed that NGF promoted the trafficking of TRPM8 to the plasma membrane. In the presence of NGF, lysosome-associated membrane protein-2 (LAMP-2) was localized to TRPM8-positive dot-like and linear structures, the latter of which were observed in the periphery of the cytoplasm. It was inferred that LAMP-2 was involved in the vesicular transport of TRPM8. Pharmacological blockade of the proteasome with MG132 led to a further increase in NGF-induced TRPM8 expression, indicating that the proteasome system played a pivotal role in the degradation of TRPM8. Our findings provide novel insight into the signaling pathways involved in NGF-mediated TRPM8 upregulation and its reversion to the normal state.

High-mobility group box 1 is an important mediator of microglial activation induced by cortical spreading depression.

Single episodes of cortical spreading depression (CSD) are believed to cause typical migraine aura, whereas clusters of spreading depolarizations have been observed in cerebral ischemia and subarachnoid hemorrhage. We recently demonstrated that the release of high-mobility group box 1 (HMGB1) from cortical neurons after CSD in a rodent model is dependent on the number of CSD episodes, such that only multiple CSD episodes can induce significant HMGB1 release. Here, we report that only multiple CSD inductions caused microglial hypertrophy (activation) accompanied by a greater impact on the transcription activity of the HMGB1 receptor genes, TLR2 and TLR4, while the total number of cortical microglia was not affected. Both an HMGB1-neurtalizing antibody and the HMGB1 inhibitor glycyrrhizin abrogated multiple CSD-induced microglial hypertrophy. Moreover, multiple CSD inductions failed to induce microglial hypertrophy in TLR2/4 double knockout mice. These results strongly implicate the HMGB1-TLR2/4 axis in the activation of microglia following multiple CSD inductions. Increased expression of the lysosomal acid hydrolase cathepsin D was detected in activated microglia by immunostaining, suggesting that lysosomal phagocytic activity may be enhanced in multiple CSD-activated microglia.

Alterations in the threshold of the potassium concentration to evoke cortical spreading depression during the natural estrous cycle in mice.

Cortical spreading depression (CSD) has been implicated in a variety of neurological disorders. However, the relationship between serum sex hormones and susceptibility to the development of CSD in naturally estrous cycling female animals is largely unknown. The natural estrous cycle of mice consists of four stages, namely, proestrus, estrus, metestrus and diestrus. We measured the serum concentration of estradiol and progesterone in estrus and diestrus and compared the minimum potassium concentrations necessary to evoke CSD in each stage and in males. In diestrus, the minimum potassium concentration required to evoke CSD was significantly lower compared to the other three phases and male animals. The serum level of estradiol is significantly higher and serum level of progesterone is significantly lower in diestrus compared to estrus. Furthermore, when we administered an estrogen receptor antagonist, the susceptibility to the development of CSD was decreased. Conversely, the administration of a progesterone receptor antagonist increased the susceptibility to CSD. Our results demonstrated that neuronal excitability related to CSD induction differs among the natural estrous phases in mice.

Repetitive trigeminal nociceptive stimulation in rats increases their susceptibility to cortical spreading depression.

We examined the ability of trigeminal nerve activation to induce cortical spreading depression in rats. Capsaicin was injected into the bilateral plantar or whisker pad for either 4 or 6 days in rats. The number and duration of cortical spreading depressions induced by potassium were significantly increased in animals injected with capsaicin in the bilateral whisker pad compared with animals injected in the bilateral plantar or in controls, while administration of a GABAA receptor agonist decreased these effects. Repetitive nociceptive stimulation of the trigeminal nerve lowers the threshold for the induction of cortical spreading depression by altering GABAergic neuronal activity.

Temporal profiles of high-mobility group box 1 expression levels after cortical spreading depression in mice.

INTRODUCTION: Cortical spreading depression (CSD) has recently been shown to induce the release of the nuclear protein termed high-mobility group box 1 from neurons, causing activation of the trigeminovascular system. Here, we explored the effects of single and multiple cortical spreading depression inductions on high-mobility group box 1 (HMGB1) transcriptional activity relative to high-mobility group box 1 protein expression levels and intracellular localization in cortical neurons and astrocytes. METHODS: Single or multiple cortical spreading depression inductions were achieved by KCl application to the mouse cerebral cortex. The animals were sacrificed at 30 minutes, 3 hours and 24 hours after cortical spreading depression induction. High-mobility group box 1 expression levels were explored with in situ hybridization, Western blotting and immunostaining. RESULTS: Cortical spreading depression up-regulated high-mobility group box 1 transcriptional activity in neurons at 3 hours in a manner that was dependent on the number of cortical spreading depression inductions. At 24 hours, the high-mobility group box 1 transcriptional activity had returned to basal levels. Cortical spreading depression induced a reduction in high-mobility group box 1 protein expression at 3 hours, which was also dependent on the number of cortical spreading depression inductions. Following cortical spreading depression, the release of high-mobility group box 1 from the nucleus was observed in a small proportion of neurons, but not in astrocytes. CONCLUSION: Cortical spreading depression induced translocation of high-mobility group box 1 from neuronal nuclei, driving transcriptional up-regulation of high-mobility group box 1 to maintain protein levels.